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Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Agric. Food Chem. 2024, 72, 10, 5358-5367
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Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V–F CRISPR/Cas Nuclease AsCas12f1

  • Hui-Min Hua
    Hui-Min Hua
    College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
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  • , 
  • Jia-Feng Xu
    Jia-Feng Xu
    College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
    More by Jia-Feng Xu
  • , 
  • Xue-Shuang Huang*
    Xue-Shuang Huang
    Hunan Provincial Key Laboratory for Synthetic Biology of Traditional Chinese Medicine, Hunan University of Medicine, Huaihua 418000, China
    *Email: [email protected]
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  • , 
  • Andrei A. Zimin
    Andrei A. Zimin
    G.K. Scriabin Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino 142290, Russia
    More by Andrei A. Zimin
  • , 
  • Wen-Fang Wang*
    Wen-Fang Wang
    College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
    *Email: [email protected]
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  • , and 
  • Yin-Hua Lu*
    Yin-Hua Lu
    College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
    *Email: [email protected]. Tel.: +86 21 64322208.
    More by Yin-Hua Lu
    Orcidhttps://orcid.org/0000-0001-5696-4018
Cite this: J. Agric. Food Chem. 2024, 72, 10, 5358–5367
Publication Date (Web):March 1, 2024
https://doi.org/10.1021/acs.jafc.3c09101
Copyright © 2024 American Chemical Society
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    Abstract

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    Genome editing tools based on SpCas9 and FnCpf1 have facilitated strain improvements for natural product production and novel drug discovery in Streptomyces. However, due to high toxicity, their editing requires high DNA transformation efficiency, which is unavailable in most streptomycetes. The transformation efficiency of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1 was significantly higher than those of SpCas9 and FnCpf1 in tested streptomycetes, which is due to its small size and weak DNA cleavage activity. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion and 46.7 and 40% efficiency for simultaneous deletion of two genes and two gene clusters, respectively. AsCas12f1 was successfully extended to Streptomyces hygroscopicus SIPI-054 for efficient genome editing, in which SpCas9/FnCpf1 does not work well. Collectively, this work offers a low-toxicity, high-efficiency genome editing tool for streptomycetes, particularly those with low DNA transformation efficiency.

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    This material is available free of charge on the ACS Publications Web site. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.3c09101.

    • Plasmid construction procedure; strains, plasmids, and primers used in this study; and schematic drawing, conjugation efficiency data, colony PCR results, and DNA sequencing maps (PDF)

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